GB2127023A - Synthesis of 25-hydroxyvitamin d2 and related compounds - Google Patents

Description

1 GB 2 127 023 A 1

SPECIFICATION Process for preparing hydroxyvitamin D, compounds and intermediates used therein

This invention relates to a process for the preparation of hydroxylated derivatives of vitamin D21 and to novel intermediates used in this process, especially 25-hydroxyvitamin D2 and the 24-epimer 5 thereof, certain alkyl and aryl-analogs thereof, 5,6-trans- and acyl derivatives of these compounds.

The D vitamins are very important agents for the control of calcium and phosphate metabolism in animals and humans, and have long been used as dietary supplements and in clinical practice to assure proper bone growth and development. It is now known that the in vivo activity of these vitamins, specifically of viamin D2 and D, is dependent on metabolism to hydroxylated forms. Thus, vitamin D2 undergoes successive hydroxylation reactions to its active forms, being first converted to 25-hydroxyvitamin D2 (25-OH-D,) and then to 1,25-dihydroxyvitamin D, (1,25-(OH),D,).

These hydroxylated forms of vitamin D2 are, because of their potency and other beneficial properties, highly desirable dietary supplements, or pharmaceutical agents, for the cure or prevention of bone or related diseases.

Whereas all metabolites of vitamin D, have been prepared by chemical synthesis, there has been 15 little work on the preparation of vitamin D2 metabolites. The known synthetic processes for the metabolites of the D,-series (especially side chain hydroxylated compounds) are, of course, in general not suitable for the preparation of the corresponding vitamin D2 metabolites, since the latter are characterized by a side chain structure (i.e. presence of a double bond and an extra methyl group) which requires a different synthetic approach from that applicable to side chain hydroxylated D, compounds.

Two compounds structurally related to 25-OH-D, have been prepared, namely 22-dehydro-25 hydroxycholecalciferol, which may be considered a 24-clesmethyl analog of 25-OH-D, (see U.S. Patent 3,786,062), and 24,2 5-di hydroxyvita min D2, the 24-hydroxy-analog of 25- OH-D2 [Jones et al.

Biochemistry 18, 1094 (1979)]. However, the synthetic methods proposed in these reports are not 25 applicable to the preparation of 25-OH-D, itself.

A novel and convenient synthesis of 25-hydroxylated vitamin D2 compounds has now been developed according to the present invention. This synthesis provides 25- hydroxyvitamin D2 (25-OH D2) and its 24-epimer, 25-hydroxy-24-epi-vitamin D2 (25-OH-24-epi D2)1 characterized by the structures shown below (where X, and X2 are hydrogen and Y is methyl), 0 25-OWD2 25-0H-24-epi D2 as well as the corresponding alkyl or aryl analogs, where Y is alkyl or aryl, and the hydroxy-protected derivatives of these compounds, where at least one of X, and X, is acyl.

In addition, the present process provides the corresponding 5,6-transisomers, and novel intermediates that are of utility for the preparation of 25-OH-D2-analogs and/or isotopically-labeled 35 products.

The term "acyl", as used herein, signifies an aliphatic acyl group of from 1 to, say, 6 carbons, in all possible isomeric forms (e.g. formyl, acetyl, propionyl, butyryl, isobutyryl and valeryl), or an aromatic acyl group (aroyl group) such as benzoyl, the isomeric methylbenzoyls and the isomeric nitro- or halo- benzoyls, or a dicarboxylic acyl group of from 2 to 6 atoms chain length, i.e. acyl groups of the type 40 ROOC(CH2)nCO-, or ROOCCH2_0-CH2CO_1 where n is from 0 to 4 inclusive and R is hydrogen or an alkyl radical, such as oxalyl, malonyl, succinoyl, glutaryl, adipyl, cliglycolyl. The term "alkyl" denotes a lower alkyl group of 1 to 6 carbons in all possible isomeric forms, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec.-butyl and pentyl, and the word "aryl" signifies a phenyl or substituted phenyl group, e.g. alkylphenyl or methoxyphenyl.

The overall process developed for the preparation of the above compounds may be divided into two general phases, namely (a) addition of a side chain fragment to a suitable steroidal precursor to produce a 5,7-diene steroid as the central intermediate, and (b) conversion of this 5,7-diene to the vitamin D structure with, as required, further modification of the side chain to produce the desired 25 hydroxylated compounds. This general scheme allows for some flexibility in the choice of specific starting material and in the exact order of individual process steps, two features that are of 2 GB 2 127 023 A 2 considerable practical advantage and convenience. According to the present invention there is provided a process for preparing a compound having the formula 1 1 Xtoe, ox, wherein X, and X2 are each independently hydrogen or acy], and Y is alkyl or aryl, which comprises 5 subjecting a 5,7-diene steroid of the formula to the following steps, (a) irradiation with ultraviolet light to obtain the corresponding previtamin D, (b) isomerization of previtamin D in an inert solvent at a temperature from about room 10 temperature to about 1001C, thereby obtaining a compound of the formula 1 )(j ol( - 411 ' >, 1 c) 0 L-i (;j i (c) acid catalyzed hydrolysis to remove the ketal protecting group, and (d) subjecting the de-protected ketone to alkylation with a reagent of the formula YMg-Hal or YLi, where Y is alkyl or aryl and Hal is chlorine, bromine or iodine, in any order provided that step (a) precedes step (b) and step (c) precedes step (d), and, optionally, (e) acylating one or both free hydroxy 15 groups.

The overall reaction sequence is illustrated by Process Scheme 1, whereas Process Scheme 11 illustrates some of the options available for executing the last four steps of the synthesis.

Starting materials for the present process are steroidal 22-aldehydes in which the ring B double bond(s) is (are) protected in an appropriate manner. As shown in Process Scheme 1, suitable compounds are for example, the PTAD-diene-protected-22-aidehyde (a) (where PTAD refers to the phenyitriazoline-3,5-dione-protecting group shown) or the 3,5-cyclo-22aldehyde (4) wherein the A' double bond is protected via i-ether formation. Both of these compounds are known products (see for example Barton et al J. Chem. Soc. (C) 1968 (197 1); and U.S. Patent 2, 623,052) and both can be carried through the steps of the present process in a basically analogous fashion.

The first step of this process comprises the addition of a suitable side chain fragment. Thus, condensation of aldehyde (1) with a sulfonVI-side chain fragment as shown in the Scheme (sulfone A, further described below) present in the form of its anion, typically in an ether or hydrocarbon solvent, provides the hydroxy-sulfone intermediate (2). The anion of the sulfone A side chain fragment can be generated by treatment of the sulfone with a strong base, such as lithium diethylamide, n-butyl lithium or ethyl 30 magnesium bromide (or similar Grignard reagent) in an ether or hydrocarbon solvent, and to this solution the steroid aldehyde (1) as an ether or hydrocarbon solution is then added. The reaction is conducted advantageously at about room temperature, and preferably under an inert atmosphere.

The analogous addition of sulfone A to aldehyde (4) provides the hydroxysulfone intermediate (5) in Process Scheme 1.

a 3 GB 2 127 023 A 3 Process Scheme I- C140 Ace, 0 P.

(1) 1 NO 0 1111-h 14 0 Wrone A 0 A i.

ACO N W" ph (2) 15< 0 40 (3) L (7) 0 1HO H0h c 7 yo (10) #6 d Km Or"3 (4) 4, 0 o SCI, 1 (W 1-4, ' b7:j d k (6) AR ---gow- 1 wi - (9) flo (60) c' 1 CH W) o (24 5) b (24 R) The next step comprises the removal of the hydroxy- and phenylsulfonyl groups in the side chain with formation of the 22(23)-trans-double bond. Thus, treatment of compound (2), in methanol solution saturated with NaHPO, with sodium amalgam under an inert atmosphere, gives compound (3) featuring the desired trans-22-double bond in the side chain. The analogous treatment of compound (5) gives the 22-olefinic compound (6). If desired, the 22-hydroxy group in compounds (2) or (5) can be acylated or sulfonylated (e.g. mesylated) prior to the Na/Hg-reduction step, but this is not generally required.

It is to be noted that, as shown in Process Scheme 1, addition of the side chain fragment, sulfone 10 A, to the alclehydes (1) or (4), does not cause epimerization at the asymetric center of carbon 20, i.e. the stereochemistry at that center is retained, as is required. If desired, retention of stereochernistry at carbon 20 may be checked at this stage by the conversion of intermediate (3) or (6) back to the aldehyde starting material. For example, subjecting compound (6) to ozonolysis with reductive work- up, using fully conventional and standard conditions, yields the corresponding C-22-aldehyde, i.e. the 15 4 GB 2 127 023 A aldehyde of structure (4). Spectroscopic and chromatographic comparison of the aldehyde obtained from ozonolysis with the original starting material confirms retention of C-20 stereochemistry.

The third operation of the process involves conversion of these ring Bprotected steroids to the desired 5,7-diene intermediate (7). In the case of the PTAD-diene-protected compound (3), this conversion can be accomplished in a single step with a strong hydride reducing agent (e.g. LiAll-1,) in an ether solvent at reflux temperature. Compound (7), can also be produced from the i-ether derivative (6), in several conventional well-known steps. The i-ether (6) may first be solvolyzed in glacial acetic acid at reflux for, say, 2 hours to yield the corresponding 5-ene3-acetate derivative (6a), which, in a hydrocarbon solution (e.g. hexane) at reflux temperature preferably under an inert atmosphere, is then treated with a brominating reagent (e.g. 1,3-di b romo-5,5-di methyl hyd anto in) over, say, 20 minutes, 10 and the resulting C-7-bromo-intermediate directly dehydrobrominated by dissolution in xylene and treatment with a base (e.g. s-collidine) at reflux temperature under an inert atmosphere, typically for about 90 minutes. The resulting product, the 5,7-diene-3-acetate can then be isolated in the usual way and purified by high performance liquid chromatography or thin layer chromatography on silica gel plates. Simple hydrolysis of the acetate (5% KOH in MeOH) then provides 5, 7-diene (7). This hydrolysis 15 step may, however, be omitted since both the 5,7-diene-3-ol (7) or the corresponding 3-0-acylates can be used for the subsequent steps of the process. Any such 3-0-acylates are, of course, also readily accessible by simple acylation of (7) according to conventional procedures.

Conversion of 5,7-diene (7) to the final vitamin D products comprises a sequence of four steps, the precise order of which may be altered as convenient. The sequence shown in Process Scheme 1 involves first the irradiation of a, typically ether or hydrocarbon, solution of the 5,7-diene (7) with ultraviolet light to yield the previtamin analog (8) which by standing at room temperature or warming (e.g. 50-90IC) in a suitable solvent (e.g. ethanol, hexane) undergoes isomerization to the vitamin D2 analog (9). The next step, removal of the ketal protecting group, is a critical one, since ketal removal by hydrolysis to give the corresponding keto-derivative (10) must be accomplished without isomerization of the 22(23)-double bond to the conjugated 23(24)-position. Isomerization of a PA-unsaturated ketone to the conjugated a,p-unsaturated ketone can readily occur on ketal hydrolysis, but must be avoided. Ketal removal can be achieved by heating ketal (9) in hydroxylic solvent under acid catalysis for, say, 1-2 hours. (it is desirable to monitor the progress of reaction by periodic chromatographic analysis of the crude reaction mixture. Analysis by HPLC is suitable and convenient.) The product, 30 ketone (10) thereby obtained, is then alkylated in the final step by means of a Grignard reagent (an alkyi-, or aryl-magnesium halide, e.g. methyl magnesium bromide in this case) to give the 25-hydroxy vitamin D2 compound (11). Alkylation via an alkyllithium reagent, e.g. methyl lithium, is also effective and convenient. If the side chain fragment, sulfone A, as used in the first step, is racemic, i.e. exists as a mixture of its (R) and (S)-enantiomers, then compound (11) will be obtained as a mixture of two C-24 epimers, i.e. (24S)-epimer (1 1a) which corresponds to the natural product, and the (24R)-epimer (1 1b) which is 25-0H-24-epi-D2. These C-24-epimers are conveniently separated by high performance liquid chromatography (HPLC) on an efficient microparticu late silica gel column to obtain 25-OWD2 (1 1a) and 25-0H-24-epi-D2 (1 1b) in pure form.

It is to be noted that with racemic sulfone A as the side chain starting material, the early synthetic 40 intermediates, e.g. (3) [or (6), and (6a)l as well as the 5,7-diene (7) and subsequent intermediates (8), (9), and (10) also occur as the two C-24-epimers. If desired and convenient, separation of epimers can be conducted at any of these intermediate stages, and the (24R) and (24S) epimers may then be processed separately through the remaining steps to yield 25-OWD2 (1 'a) or 25-0H-24-epi-D2 (1 1b) as desired. It is generally convenient to effect separation of the final products.

Since the mixtures of (24R)- and (24S)-epimers arise when the side chain fragment, sulfone A is racemic, it is also possible, if desired, to circumvent the need for epimer separation by the use of optically active sulfone A. Thus, use of the (R)-epimer of sulfone A in the present process yields specifically 25- OWID2 (1 la), while use of the corresponding (S)-epimer of sulfone A provides 25-OW 24-epi-D, (1 1b) as well as, of course, the respective intermediates in the pure (24R) or (24S) forms; the use of such optically pure sulfone starting material requires no other modification of the process steps.

It is also important to note that the exact sequence of steps between the 5,7-diene (7) and the final products may be altered. Indeed, there are three convenient synthetic sequences, all involving the same steps, but in different order. These alternate sequences are shown in Process Scheme 11, where X, and X2 in the structural drawings signify hydrogen or an acyl group such as acetyl, propiony], butyryi, 55 benzoyl or substituted benzoyl. The first sequence (identified by the letter A in Process Scheme 11), leading from diene (M) (which, when X,=H, corresponds to diene (7) of Process Scheme 1) to intermediates (BA), (9A), and to the final product (11) presents the order of reactions as discussed above. 60 Alternatively, the ketal in diene (7A) may be hydrolyzed first (see sequence B), to yield the diene- 60 ketone identified as (78) in the scheme, which after irradiation gives the previtamin ketone (88), and thermal isomerization then leads to vitamin D2-ketone (10), which via a final Grignard reaction yields the 25-0H-D,- epimers (11). In the third sequence (C), the 5,7-diene-ketone (78) is first reacted with Grignard reagent to yield 65 the 25-hydroxy intermediate (7C), which after irradiation gives the corresponding 25-01- 1-previtamin D2 65 4 GB 2 127 023 A WC), and a final thermal isomerization provides the 25-OH-D2 products (11).

Thus, these three sequences differ only in the exact order in which specific steps are carried out, but the experimental conditions for the individual steps are analogous to the procedures described earlier, and as described in detail by the Examples. Of these three, sequence A is generally preferred, because of the utility of intermediates such as (9A) and (10) for the preparation of other vitamin D2- 5 analogs and/or labeled derivatives (see also below).

For any of these sequences, the 5,7-diene (7) may be used as the free hydroxy compound or as its C-3-acylate. Depending on the subsequent reaction sequence, the final 25- OH-D2 products will be obtained as the free hydroxy compounds or, if desired, as the C-3-, or C- 25-acylates, or 3,25 diacylates. Thus, synthesis according to sequence A or B would normally provide the 25-OH-D2 products as the free hydroxy compounds since the final Grignard reaction common to both sequences removes any acyl groups. Sequence C can be used to produce the 25-OH-D, epimers (11) as the free hydroxy compounds, or as the 3- or 25-monoacylate, or 3,25-diacylate, depending on the intermediate used. For example, the 5,7-diene intermediate (7C) shown in Process Scheme 11, may be used as the 3- acyl, or 25-acyl, or 3,25-diacyl derivatives, which are avaifable from the 3,25-diol by re ction ith acyl15 chloride or acid anhydride reagents according to conventional procedures. Thus, reaction of 3,25-diol intermediate (7C) with acetic anhydride is pyridine at room temperature gives the 3-acetate, and the corresponding 3, 25-diacetate can be obtained byfurther acylation at elevated temperature; the latter may be selectively hydrolyzed with dilute KOH/MeOH at room temperature to give the 25-monoacetate. Further conversion of any of such acyl intermediates through the remaining steps [to (8C) and 20 U 01 in Process. Scheme 11, then yields the 25-OH-D2-epimers (11) in any desired acylated form.

Process Scheme 11 0 0 B X80..

A 0 Li a no=(BA) (M) 1 B 0 M MB) --C--- -'d "ox, X40 (LC) 1 c 1 10 nc) (pc) -'h " X2 A 1 B i c 4e c 0 OX2 A A X OX01 X'oo& (9A) (10) (LI) Process Scheme Hf 5401-JY Iso._ly 0 Pb-5---% 0 D 0 1 0 0 0 0 sultone A The individual 25-0H-D2-epimers, 25-OWD2 (1 1a) or 25-0H-24-epi-D, (1 1b) when obtained in the free hydroxy forms, are also conveniently acylated at the C-3, or C-25, or at both positions, by reaction with acid anhydrides or acyl chlorides using conventional conditions. Thus, 25-OWD2 (1 l a) may be acylated to yield, for example, the 25-01-1-D2-3-acetate, or the corresponding 3,25-diacetate.

6 GB 2 127 023 A The 3-monoacetate, in a like fashion, may be further acylated at C-25 by treatment with a different acylating reagent, or, alternatively, the 3,25- diacetate may be selectively hydrolyzed by mild base (KOH/MeOH) to give the 25-monoacetate, which if desired can be reacylated with a different acyl group at C-3. In addition to acetic anhydride, suitable acylating agents include propionic, butyric, pentanoic or hexanoic anhydrides or the corresponding acid chlorides, or atomatic acylating agents such as the acid chlorides of benzoic or substituted benzoic acids, or the anhydrides of dicarboxylic acids, such as succinic, glutaric, adipic, diglycolic anhydrides, or the acyi chlorides of these dicarboxylic acid monoesters.

In addition to the acylates, the 5,6-trans-isomers of 25-OH-D2 and 25-OH24-epi-D2 are compounds of potential utility in medical applications because of their considerable vitamin D-like activity. These 5,6-trans-compounds can be prepared from the 5,6-cis-isomers (i.e. 1 la or 1 1b) by iodine catalyzed isomerization according to the procedures of Verloop et aL Rec. Trav. Chim. Pays Bas 78, 1004 (1969) and the corresponding 3- and/or 25- acylates can likewise be obtained by analogous isomerization of the corresponding 5,6-cis-acylates, or by acylation of the 5,6-trans-25-OH-D compounds.

It is to be noted also that the 25-keto-intermediate (compound (10) in Process Scheme 1) can serve as a substrate for a convenient preparation of 25-OH-D, or its 24- epimer in isotopically labeled form, namely by reaction of the ketone with commercially available isotopically labeled Grignard or methyl lithium reagents to provide 25-OH-D2 compounds labeled at carbon 26 with 13C, 14C, 2 H or 3 H.

Furthermore, the keto-vitamin D compound U 0) also serves as a convenient intermediate for the 20 synthesis of 2 5-OH-D.-a na logs, of the formula (12), X,01 1, Dic. k Y 1 1 (a) - Q1 r where X, and X2 are independently hydrogen or acyl, and Y is an alkyl group other than methyl or an aryl group. These compounds can be prepared by reaction of ketone (10) with the appropriate alkyl- or aryl- Grignard or alkyl- or aryl-lithium reagent. For example, treatment of ketone (10) with ethyl magnesium iodide yields product (12), where X2X2=H, and Y=ethyl; likewise treatment of ketone (10) with isopropyl magnesium bromide, or phenyl magnesium bromide yields the corresponding 25OH-D2- congeners of structure (12) above, where Y=isopropy], or phenyl, respectively, and other alkyl analogs of structure (12), e.g. where Y is propy], butyl, sec.-butyi, isobuty], pentyl, can be prepared by analogous reactions. Acylation of these products by the procedures discussed above provides the C-3-, or C-25-0-acylates, or 3,25-di-O-acylates, and isomerization of the 5,6- double bond according to the procedure of Verloop et al. cited above yields the 5,6-trans-isomers of the compounds of structure (12) and/or the acylates thereof.

Since the compounds where Y is a higher homolog of methyl, are generally more lipophilic, the aikyi- or aryl-analogues represented by structure (12) above or their 5,6- trans-isomers, are expected to 35 have utility in applications where a greater degree of lipophilicity is desired.

The required side chain fragment, sulfone A, can itself be prepared according to the process shown in Process Scheme Ill. This straightforward synthesis involves, as a first step, the reaction of commercially available 4-hydroxy-3-methyi-butan-2-one with p- toluenesulfonylchloride to form the corresponding toluenesulfonyl ester. This product is then treated with thiophenol in the presence of 40 base (e.g. potassium t-butylate) whereby the toluenesulfonyl group is displaced and the corresponding phenylthioether is formed. In the next step, the ketone group is protected as the ethylene ketal by reaction with ethylene glycol under acid catalysis, using conventional conditions. Oxidation of this product with a peracid (e.g. perbenzoic acid, or m-chloroperbenzoic acid) in halocarbon solution (e.g.

CH2C12) then provides the desired sulfone A.

If sulfone A is desired in optically active form, i.e. as the pure (R) or (S)-epimer, it is appropriate to use optically active starting materials, for example the ethylene ketal of (3R)-4-hydroxy-3-m ethyl butan-2-one or the ethylene ketal of (3S)-4-hydroxy-3-methyibutan-2-one. Each of these ethylene ketals is then processed through the steps of Process Scheme Ill to yield from the (R) ketal starting material the 0-enantiomer of sulfone A, and from the 0-ketal the 0- enantiomer of sulfone A. The 50 (R) and (S) ketal starting materials are themselves conveniently obtained from commercially available racemic a-m ethyl acetoa cetate ethyl ester (ethyl 2-methyi-3-oxo- butonate) as follows: The keto ester is converted to the ethylene ketal ester by reaction with ethylene glycol under acid catalysis using conventional procedures, and the ester function is then reduced (LiA]H, in ether) to yield the racemic ketal-alcohol (2,2 -ethyl en edioxy-3-m ethyl buta n-4-ol). Resolution of the racemix mixture can be accomplished by conversion to a mixture of diasteromers (by reaction of the alcohol function with an optically active acylating agent) which are then separated. For example, the alcohol can be converted z 7 GB 2 127 023 A to the corresponding a-methoxyatrifluoromethylphenylacety1 derivative (or similar optically active acylate) by reaction in pyridine solution with the chloride of optically active (+) ce-methoxy-a-trifluoromethylphenylacetic acid (according to the procedures of, for example, Dale et aL, J. Org. Chem. 34, 2543 (1961); Eguchi etaL, P roc. Nati.Acad. Sci. USA 78, 6579 (1981W this disastereomeric acyl- derivative mixture is now separable by HPLC or similar chromatographic methods into its two components, namely the acylate of the (R)-enantiomer and the acylate of the (S)-enantiomer. Removal of the acyl group in each compound by base hydrolysis under standard conditions then provides the ethylene ketal of (3R)-4-hyd roxy-3 -methyl buta n-2-on e, and the ethylene ketal of (3S)-4-hydroxy-3methylbutan-2-one, which are then separately processed to the respective sulfone A anantiomer as described above. If desired, optically active hydroxybutanone intermediates, i.e. QR)-4-hydroxy-3methyl butan-2-on e and (3S)-4-hydroxy-3-methyibutan-2-one, can also be prepared from naturally occurring optically active substrates. Thus, by reaction of the known (S)-3-hyd roxy-2-m ethyl p ropa no ic acid (phydroxyisobutyric acid) with methyl lithium there is obtained QS)-4-hyd roxy-3-m ethyl buta n-2 one; and the corresponding (3R)-hydroxybuta none can be prepared from the same (S)-hydroxy-iso- butyric acid, by the transposition of functionalities, i.e. elaboration of the hydroxymethyl group to a 15 methyl ketone function, and reduction of the acid to alcohol, according to obvious and conventional procedures.

The present invention is further described by means of the following Examples, in which numerals designating specific products (e.g. compounds (1), (2), (3), etc. in Examples 1 through 12, or compounds (7A), (88), (8C), etc. in Examples 13 and 14) refer to the structures so numbered in 20 Process Schemes 1 or 11.

Example 1

The C-22 aldehyde (1) is obtained by degradation of ergosterol acetate (in which the ring B diene system has been protected by Diels-Alder addition of 4-pheny]-1,2,4- triazoline-3,5-dione) according to the proccedure of Barton et al. (supra). The i-ether aldehyde (4) is obtained from stigmasterol by the 25 method of U.S. Patent 2,623,052.

Example 2

Synthesis of the side chain fragment (sulfone A) To a stirred solution of 4-hydroxy3-methylbutan-2-one (12.75 g; 0. 125 mol) in pyridine (100 mi) is added p-toluenesulfonyl chloride (p-TsCO (33.25 g, 0.175 mol) in portions, and after standing for 30 14 h at room temperature, the reaction mixture is poured into water and extracted with CH,Cl, The extract is washed several times with aqueous CuS04 solution and water and then dried over anhydrous sodium sulfate. Removal of solvent under reduced pressure gives the crude tosylate which is used directly for the next reaction.

Thiophenol (14 g) dissolved in DMF (100 mi) is treated with T-BuOK (14 g). To this reagent, the 35 tosylate is added and after 12 h at room temperature, the reaction mixture is poured into water and extracted with CH,Cl, The extract is washed with aqueous Na,CO, solution and water, then dried.

Evaporation of solvent gives an oily residue which is purified by silica gel column chromatography. Pure phenyl suffide is eluted with benzene (yield 15 g).

To this phenyl sulfide derivative (15 g), in benzene (100 m]), ethylene glycol (6 g) and p-TsOH (20 40 mg) is added and the reaction mixture is heated under a Dean-Stark trap for 3 h. After cooling, it is extracted with Na2C03 solution and water, then dried and the solvent is evaporated. The product, the desired ketal, is chromatographically homogenous and can be used in the next step without further purification.

Crude ketal in dichloromethane (250 mi) solution is treated with mchloroperbenzoic acid (m CPBA) (80-85%, 27 9, added in portions) while maintaining the temperature of the reaction mixture below 30cl C. After the addition of reagent, the reaction is allowed to stand at room temperature with occasional shaking. When the reaction reaches completion (about 1.5 h), the aromatic acids are removed by extraction with aqueous N1H, and the organic layer is washed with water and dried.

Evaporation of solvent gives the oily sulfone (sulfone A) in essentially quantitative yield (19 g). The product is substantially pure (homogenous by TILC) and can be used with any further purification; 'H-NMR; 8; 1. 18 (d, J=7 Hz, 3H, CH,-CH-), 1.19 (s, 3H, CH,-C-), 3.84 (m, 4H, ketal-H), 737.6 and 7.6-7.9 (m, 3H+2H, aromatic protons); IR, v rn K'r: 1305, 1147, 1082 cm-'; mass spectrum, mIz ax (rel. intensity): 255 (M±Me, 21), 184 (66), 87 (92), 43 (100).

Example 3 Coupling of sulfone A to aldehyde (l): hydroxysulfone (2) and olefin (3).

Grignard reagent is prepared from Mg (535 mg; 22.22 mmol) and ethyl bromide in ether (10 mi), and the vigorously stirred solution is treated with sulfone A (6 g; 2.22 mmol) in benzene (6 mi). The precipitate formed is ground with a spatula, stirring is continued, and after 15 min the aldehyde (1) (2.0 g) is added in benzene (10 m]). The reaction mixture is stirred at room temperature for 24 h, then poured into aqueous (NHIS04solution and extracted with benzene. The organic layer, after washing with water, drying and evaporation gives an oily residue which is chromatographed on silica gel. In the 8 GB 2 127 023 A 8 benzene-ether fractions (82), excess sulfone is recovered (4.5 g); elution with benzene-ether Q:1) affords unreacted aldehyde (1) (1.0 g); the reaction products (2) are eluted with ethyl acetate.

The crude mixture of steroidal a-hydroxysulfones (2) is dissolved in methanol (200 mi) saturated with Na2HPO, Sodium amalgam (5.65%, 15 9) is added and the reaction mixture is stirred at 40C for 5 15h.

After completion of the Na/Hg reduction, mercury is removed by filtration, and methanol by evaporation under reduced pressure, water is added and the organic material is extracted with benzene. After drying and evaporation of solvent, the oily residue is chromatographed on a silica gel column. Elution with benzene-ether 0:4) gives compound (3) a colorless foam; 'H-NIVIR, 8: 0.80 (s, 10 18-H),0.97(s,19-H),1.22(s,26-H), 3.93(m,4H,ketal-H),4.44(m,1H,3-H),5.25-5.45( m, 2H, 22-H and 23-H), 6.23 and 6.39 (doublets, J=8 Hz, 2x 1 H, 7-H and 6-H), 7.25-7-.45-(m, 5H, -:-C,H,); IR, v., CHC13 ax:3603 (O-H), 1749,1692 (C=O), 1406 1038 cm-l; mass spectrum, mlz: 440 (M'triazoline, 24), 87 (100).

(To increase yield, unreacted aldehyde (1), as recovered above, can be recycled through the sulfone addition, and the resulting a-hydroxy sulfones (2) are then, as above, treated with sodium 15 amalgam in buffered methanol to provide additional olefin (3). The above reactions are prefereably conducted under an inert atmosphere, such as argon.) Example 4

Coupling of sulfone A to aldehyde (4): hydroxysulfone (5) and olef in (6) Grignard reagent is prepared from Mg (75 mg, 3.1 mmol) and ethyl bromide in ether (10 m]). To 20 the stirred solution of ethyl magnesium bromide, sulfone A (891 mg; 3.3 mmol) in benzene (5 mi) is added. After stirring the resulting suspension at room temperature for 15 min, a solution of aldehyde (4) (290 mg) in benzene (5 mO is added. The reaction is continued for 2.5 h, then quenched with saturated (NHIS04 solution (5 mi) and diluted with ether. The separated organic layer is washed with water, dried, and evaporated. The oily residue containing (5) is treated with acetic anhydride (2 m[) and 25 pyridine (2 mi). The reaction mixture is allowed to stand for 24 h, poured into water and extracted with benzene. The benzene extract is washed with an aqueous solution of CuSO4, water, dried, and evaporated. The crude product [the acetate of (5)l is dissolved in methanol saturated with Na2HP04 and sodium amalgam (5.65%, 8 g) is added. The reaction mixture is stirred at 41C for 16 h. After the reaction, mercury is removed by filtration, methanol is evaporated, and water and benzene are added to 30 dissolve the residue. The benzene layer is dried and evaporated. The oily residue is chromatographed over silica gel. Elution with benzene-ether mixture (93:7) affords compound (6) (206 mg; 54%), 'HNIVIR, 8: 0.74 (s, 18-H), 1.04 (s, 19-H), 1.25 (s, 26-H), 2. 78 (m, 1 H, 6-H), 3.34 (s, 3H, -OCH), 3.97 (m, 4H, ketal-H), 5.2 5-5.45 (m, 2H, 22-H and 23-H), 1 R,.. KBr: 3470 (O-H), 1095 cm-'; mass ax 5) spectrum, mIz (rel. intensity): 456 (M', 1), 441 (M'-Me, 4, 87 (100). It should be noted that the ackylation step described above is not essential and may be omitted if desired; i.e. the hydroxysulfone (5) may be submitted directly to Na/Hg-reduction, as in Example 3. The above reactions are preferably conducted under an inert atmosphere, e.g. argon.

Example 5

Removal of PTAID-protecting group: 5,7-diene (7) A mixture of the compound (3) (1 g) and lithium aluminum hydride (1.8 g) in THF (120 ml) is heated under reflux for 10 h. After cooling, excess reagent is destroyed with a few drops of water, and the mixture is dried over anhydrous MgSO, filtered, and solvent is evaporated to give colorless crystalline material. Crude diene 7 is repeatedly crystallized from ethanol; first and second crops combined give 415 mg of (7). The mother liquor is chromatographed on silica gel column, to give with 45 benzene-ether (7:3), an additional 120 mg of (7); total yield 535 mg (79%); m.p. 132-1340C (from ethanol), IH-NMR, & 0.63 (s, 18-H), 0.95 (s, 19-H), 1.23 (s, 26-H), 3.63 (m, 1 H, 3 -H), 3.95 (m, 4H, ketal-H), 5.20-5.50 (m, 3H, 22-H, 23-H and 7-H), 5.57 (m, 1 H, 6-H); IR, v "':3430(0-H),1063, max 1038 cm-'; mass spectrum, m1z (rel. int.): 440 (M, 50), 407 (W-1-120-Me, 11), 87 (100); UV, Ama'tOH:

x 282 nm (E=1 1,000).

Example 6

Irradiation of compound (7): previtamin analog (8) A solution of diene (7) (50 mg) in 150 mi of benzene-ether W4) is cooled on ice and deoxygenated with argon for 20 min. The reaction mixture is irradiated under argon atmosphere for 18 min with a mercury arc lamp (Hanovia SA-1) fitted with a Vycor filter. The solvent is evaporated and 55 the residue is chromatographed on HPLC (6.2 mmx25 cm m icroparticu late silica gel, 4 m]/min, 1400 psi [98 kg/cm11) and eluted with 2% 2 propanol in hexane to yield 22 mg (44%) of previtamin (8); 'H NMR; 8: 0.73 (s, 18-H), 1.24 (s, 26-H), 1.64 (s, 19-H), 3.96 (m, 5H, ketal-H and 3 -H), 5.35 (m, 2H, 22-H and 23-H), 5.50 (m, 1 H, 9-H), 5.69 and 5.94 (doublets, J=1 1.5 Hz, 2 x 1 H, 6-H and 7-H); UV, Ajt: 263 nm (E=8,900).

X 9 GB 2 127 023 A 9 Example 7 Isomerization of (8) to the vitamin-analog (9) Previtamin 8 (22 mg) is dissolved in ethanol (40 mi) and heated under reflux for 150 min (argon atmosphere). The product is purified by HPLC to yield 18 mg (82%) of the pure vitamin (9); 'H-NMR, 5 8:0.75 (s, 18H), 1. 23 (s, 26-H), 3.94 (m, 5H, ketal-H and 3-H), 4.81 and 5.04 (2 narrow m, 2x 1 H, 190- and 19(E)-H), 5.33 (m, 2H, 22-H and 23-H), 6.03 (cl, J=1 1 Hz, 1 H, 7-H), 6.22 (cl, J=1 1 Hz, 1 H, 6-H); mass spectrum, mIz (ref. int.): 440 (M', 17), 87 (100), UV, Amj": 265 nm (E=1 7,000).

X Example 8 Hydrolysis of the ketal: keto-vitamin D2-analog (10) To the solution of compound (9) (18 mg) in ethanol (35 mi), p- toluenesulfonic acid (7.5 mg) in 10 water (1 mi) is added and the reaction mixture is heated under reflux for 90 min (the reaction course is monitored by HPLC). The solvent is evaporated, the residue is dissolved in benzene and extracted with water. The benzene solution is dried (anhydrous M9S04), and evaporated to yield product (10) (16 mg; 99M. 'H-NMR, 8: 0.57 (s, 18-H), 1.04 (d, J=7 Hz, 2 1- H), 1.13 (d, J=7 Hz, 28-H), 2.12 (s, 3H, 26-H), 3.10 (m, 1 H, 24-H), 3.96 (m, 1 H, 3 -H), 4.82 and 5.05 (2 narrow m, 2x 1 H, 190- and 19(E)-H), 5.25.5 (m, 2H, 22-H and 23-H), 6.03 (d, J=1 1.5 Hz, 1 H, 7-H), 6.22 (d, J=1 1.5 Hz, 1 H, 6-H), IR, A CH03:

ax 3596 (O-H), 1709 cm-1 (C=O) mass spectrum, mIz (ref. int.): 396 (M+, 41), 363 W-1-120-Me, 13), 271 (M±side chain, 16), 253 (m±side chain-H,O, 23), 136 (100), 118 (95); UV, A,,, It: 265 nm ,x (E= 17,900).

Example 9

Reaction of ketone (10) with methyimagnesium iodide: 25-OWID, (11 a), and its epimer (11 b) Grignard reagent is prepared from magnesium (240 mg) and methyl iodide in anhydrous ether (20 mi). To one-tenth of this solution (2 ml; 0.5 M solution of CH,Mgi) ketone (10) (16 mg; 0.04 mmol) in ether (2 mi) is added. The reaction mixture is stirred at room temperature for 2 h under an inert atmosphere, then quenched with aqueous solution of N1-14C1, diluted with benzene and washed with 25 water. The oragnic layer is separated, dried and evaporated. The crude product is first purified by silica gel column chromatography (elution with 20% ether in benzene) and the mixture of (1 1a) and (1 1b) (16 mg; 96%) thereby obtained is then repeatedly chromatographed on HPLC column using 2% 2 propanol in hexane as an efuent to separate the 24-stereoisomers, 24-epi- 25-0H-D2 (1 1b) and 25-01-1 D, (1 la). Chromatography and rechromatography of each stereoisomer yields 4 mg of (1 1b) (collected 30 at 68 mi), 4 mg of (1 l a) (collected at 74 mi) and 7 mg of the mixture of both epimers. Treatment of 2 mg of the epimer mixture with excess acetic anhydride in pyridine solution at room temperature overnight followed by standard work-up yields the corresponding 3-0- acetates.

25-OWD, (1 1a): [a]25+56.80 (C=0.2 in EtOH);'H-NMR, 8: 0.57 (s, 18-H), 1. 00 (d, J=7 Hz, 28 D H), 1.04 (d,J=7 Hz, 21-H), 1.15 and 1.17 (2 singlets, 26-Hand 27-H), 3.95 (m, 1H, 3-H), 4.82 and 35 5.05 (2 narrow m, 2x 1 H, 19(Z)- and 19(E)-H), 5.23-5.43 (m, 2H, 22-H and 23-H), 6.05 and 6.22 (2 doublets, J=1 1 Hz, 2 x 1 H, 7-H and 6-H); IR,An, ' ": 3401 (O-H), 1645, 1631 (C=C), 97 1, cm-1 (trans ax C=Q mass spectrum, mIz (ref. int.): 412 (M', 63),394 W-1-120,10), 379 (M± H,O-Me, 23), 271 W side chain, 37), 253 (M±side chain-1-120, 43), 136 (100), 118 (86), 59 (99) UV,.', "': 2 nm Ani x 65 (E=1 7,950).

24epi-25-0H-D2 (1 1b): [a125+50.70 (C=0.2 in EtOH), 'H-NMR, 8: 0.57 (s, 18-H), 0.99 (d, J=7 D Hz, 28-H), 1.03 (cl, J=7 Hz, 21-H), 1.14 and 1.16 (2 singlets, 26-Hand 27- H), 3.94 (m, 1 H, 3-H), 4.82 and 5.03 (2 narrow rn, 2xl H, 19(Z)- and 1 9(E)-H), 5.20-5.40 (m, 2H, 22- H and 23-H), 6.04 and 6.22 (2 doublets, J=1 1 Hz, 2 x 1 H, 7-H and 6-H), fR, v,, KBr:3401 (OH), 1643,1630 W=C), 97 1, cm-1 ax (trans C=Q mass spectrum, mIz (ref. int.): 412 (M+, 62) 394 (M±H20; 12), 379 (M'-H20-Me, 31), 45 UV, X,,1,EtOH 271 (M±side chain, 44), 253 (M±side chain-H,O, 55), 136 (100), 118 (67), 59 (38); ax: 265 nm (E=1 7,300).

It should be noted that from pure provitamin (7) further synthesis (i.e. the irradiation, isomerization, deketalization and Grignard reaction steps) may be accomplished without chromato graphic purification of any intermediate. Careful column chromatography on silica gel before the final 50 separation on HPLC removes all by-products.

By reaction of 25-OWD, (1 1a) with each of the following acylating reagents; acetic anhydride, propionic anhydride, benzoyl chloride and succinic anhydride, under conventional conditions there is obtained, respectively:

25-0H-D,-3-acetate 55 2 5-01-1 -D2-3,2 5-di acetate 25-01-1-132-3-propionate 25-0HD2-3,25-dipropionate 25-0H-D2-3-benzoate 25-0H-D,-3,25-dibenzoate 60 25-0H-D2-3-hemisuccinate.

GB 2 127 023 A 10 By reaction of 25-0H-24-epi-D2 (1 1b) with, respectively, acetic anhydride, benzoyi chloride and digiycolic anhydride, under conventional conditions, there is obtained, respectively:

2 5-01-1-2 4-epi-D,-3,2 5-di acetate 25-0H-24-epi-D2-3-benzoate 25-0H-24-epi-D2-3-hemidigiycolate.

Example 10 By coupling of aldehyde (1) with optically active 0-sulfone A having the structure p hs l,') 0 Li and subsequent Na/Hg reduction of the product according to the conditions of the experiment described in Example 3, there is obtained compound (3) having the (24S) configuration in the side 10 chain as shown by the structure, 1 A and treatment of this product with LiAM, according to the conditions of Example 5 provides the 5,7diene (7) having the (24S)-side chain configuration; by irradiation of this product and subsequent thermal isomerization according to the conditions of Examples 6 and 7 there are obtained, successively, the previtamin D compound (8) and vitamin D compound (9) having the (24S) configuration. Hydrolysis of compound (9) thus obtained, according to the conditions of Example 8, provides the (24S)-ketovitamin D compound (10) and from this product, by a Grignard reaction, according to Example 9 there is obtained 25-01-1-13, (structure (1 1a) in Process Scheme 1).

Example 11 Using optically active (S)-suifone A, having the structure 0 ph I$ 5,, A-., 1. 0 o 0 Li in the reactions described in Example 3, there is obtained compound (3), having the (24R)-side chain structure as shown below -1 C ' 0 Li and reduction of this product according to the conditions of Example 5 provides the 5,7-diene (7) having the (24R) configuration. Irradiation of (24R)-(7) according to Example 6 gives the previtamin D analog (8) with the (24R) configuration, and by subsequent thermal isomerization, according to the conditions of Example 7, there is obtained the vitamin D compound (9) having the (24R)-side chain configuration. Ketal hydrolysis, according to the conditions of Example 8, then yields the (24R)-25- 30 ketovitamin D (10), and by a reaction of this product with a methyl Grignard reagent according to the conditions of Example 9, there is obtained 25-hydroxy-24-epi-vitamin D2 (structure 1 lb, in Process Scheme 1).

Example 12

Preparations of 5,6-trans-compounds 25-OH-D, (compound 11 a) is dissolved in ether containing a drop of pyridine and treated with a solution of iodine in hexane (ca. 0.5 mg/ml) for 15 min. Addition of an aqueous solution of sodium thiosulfate, separation of the organic phase, and evaporation of solvents yields a residue, from which the desired 25-hydroxy-5,6-trans-vitamin D, is isolated by HPLC using a microparticulate silica gel column and 2% of 2-propanol in hexane as eluent.

By the same procedure, there is obtained from 25-hydroxy-24-epi-D2 the corresponding transisomer, namely 25-hydroxy-5,6-trans-24-epi-D2.

11 GB 2 127 023 A 11 From 25-01-1-D2 3-acetate, there is obtained 25-0H-5,6-trans-D, 3-acetate, and from 25-0H-24epi-D2 3-acetate there is obtained 25-0H-5,6-trans-24- epi-D2 3-acetate by the application of the above isomerization procedure.

Acylation of 25-0H-5,6-trans-D2 or 25-0H-5,6-trans-24-epi-D, under conventional conditions 5 provides the respective acylates, such as:

25-0H-5,6-trans-D2-3-acetate 2 5-0 H-5,6-trans-D,-3,2 5-di acetate 25-0H5,6-trans-D2-3-benzoate 25-0H-5,6-trans-D2-3-acetate-25-benzoate 25-0H-5,6-trans-24-epiD2-3-acetate 25-0H-5,6-trans-24-epi-D2-3,25-dibenzoate.

Example 13

Hydrolysis of 5,7-diene-25-ketal (compound (M), where X,=H) using the conditions described in Example 8 gives 30-hyd roxy-2 4-m ethyl 27-norcholesta-5,7,22-trien-25- one (compound 7B, where X,=H). Irradiation of this product under conditions analogous to those of Example 6 gives the 25-keto 15 previtamin D, analog characterized by structure (BB), where X,=H. Heating of (813) in an ethanol solution according to the conditions of Example 7 provides the 25-keto vitamin D, product (compound 10, where X,=H).

Example 14 20 Reaction of 3p-hydroxy-24-methyi-27-norcholesta-5,7,22trien-25-one (compound (78), where 20 X,=W as obtained in Example 13 with methyl magnesium bromide in accordance with the conditions of Example 9 gives 24-m ethyl cho 1 esta-5,7,22-trie ne-3p,2 5-dio 1 (compound (7C), where XlX2=H). Irradiation of this product, according to the conditions of Example 6, gives 25-hydroxy previtamin D, product characterized by structure (8C, where X1=X2=H). Thermal isomerization of this previtamin 25 using the conditions of Example 7 provides the 25-hydroxyvitamin D2 compound (11; where XlX2=H). Processing of 24-methylcholesta-5,7,22triene-3p,25-dioI 3,25-diacetate (compound 7C, X1=X2=acetyl) through the reaction steps involving irradiation and thermal isomerization according to the conditions of Examples 6 and 7 respectively gives the 25-OWD, 3,25diacetate epimers (compound (11), where XlX2=acetyi).

Example 15 Using the conditions analogous to those of Example 9, Mg is reacted with the following halides, ethyl iodide; propyl iodide; isopropyl bromide; butyl bromide; sec.-butyl iodide; isobutyl iodide; pentyl iodide; and phenyl bromide, 35 to obtain the corresponding Grignard reagents. By reaction of each of these reagents with ketone (10) by procedures analogous to that of Example 9, there are obtained, respectively, the following products:

compound (12) where X,=X,=H, Y=ethyl compound (12) where X1=X2=1-1, Y=propyl compound (12) where X,=X,=H, Y=isopropyl 40 compound (12) where X,=X2=H, Y=butyi compound (12) where X1=X2=1-1, Y=secAutyi compound (12 where X1=X2=1-1, Y=isobutyl compound (12) where X,=X2=1-1, Y=pentyl compound (12) where X1=X2=1-1, Y=phenyl 45 By reaction of ketone (10) with isotopically labeled methyl Grignard reagents, namely 13 CH3Mgl' 14 CI-13M911 C2 H3MgI1 C3 H,MgI, under conditions analogous to those of Example 9, there are obtained, respectively, the following products:

compound (12) where y='3CH3, X1=X,=H compound (12) where y=14CH, X1=X2=H compound (12) where y=C2H3, X1=X,=H compound (12) where y=C3H3, X,=X,=H characterized by isotopic substitution in the methyl group of carbon 26 of the molecule.

12 GB 2 127 023 A 12

Claims (1)

1. Claims

   1. A process for preparing a compound having the formula 1 1 Xgoc OX,L wherein X, and X2 are each independently hydrogen or acy], and Y is alkyl or aryl, which comprises 5 subjecting a 5,7-diene steroid of the formula k 1JY 1 N.

   xi CC& to the following steps, (a) irradiation with ultraviolet light to obtain the corresponding previtamin D, (b) isomerization of previtamin D in an inert solvent at a temperature from about room 10 temperature to about 1 OOOC, thereby obtaining a compound of the formula 1 xiot&- -4, ' X' ( 1 LY z 4 (c) acid catalyzed hydrolysis to remove the ketal protecting group, and (d) subjecting the de-protected ketone to alkylation with a reagent of the formula YMg-Hal or YLi, where Y is alkyl or aryl and Hal is chlorine, bromine or iodine, in any order provided that step (a) precedes step (b) and step (c) precedes step (d), and, optionally, (e) acylating one or both free hydroxy 15 groups.

   2. A process according to claim 1 wherein X, and X, are hydrogen and Y is methyl.

   3. A process according to claim 1 or 2, wherein step (c) precedes step (a), which is followed by steps (b) and (d) sequentially.

   4. A process according to claim 1 or 2 wherein steps (c) and (d) precedes steps (a) and (b). 20 5. A process according to any one of claims 1 to 4 wherein the 5,7-diene steroid is a single epimer.

   1 6. A process according to claim 1 substantially as described in the Examples. 7. A compound having the formula:

   whenever prepared by a process as claimed in any one of the preceding claims.

   13 GB 2 127 023 A 13 8. A compound having the formula:

   where N is a steroid nucleus having the formula:

   of C# m Li 01-1 X,OC&' oroftheformula:

   where acyl.

   0 X,OC represents a single or double bond, the double bond(s) of which are protected, and X, is hydrogen or 9. A compound according to claim 8 in which N has the formula:

   X, 1 1 0 0 it 0e 1: -3 0 n j 10. A compound having the formula wherein X, is hydrogen or acyl, and R is R x, OCO r Jet 4.

   0 X.2 Y wherein X2 is hydrogen or acyl, and Y is alkyl or aryl.

   11. A compound according to claim 10 in which X, and X2 are hydrogen or acetyl and Y is methyl.

   14 GB 2 127 023 A 14 12. A compound having the formula P 1 X&C 0 sI- t, wherein X, is hydrogen or acy], and R is as defined in claim 10. 13. A compound according to claim 12 in which X, and X, are hydrogen or acetyl and Y is methyl. 5 14. A compound having the formula R 1 1 X4 D X0.

   wherein X, is hydrogen or acyl, and R is as defined in claim 10 with the proviso that when Y is methyl, X, and X, cannot both be hydrogen.

   15. A compound according to claim 12 in which R is the group and X, is hydrogen, acetyl or benzoyi.

   16. A compound according to claim 14 in which R is the group cl and X, is hydrogen, acetyl or benzoyi.

   17. A compound according to claim 14 in which R is the group 0 2.

   18. A compound according to claim 17 in which X, is acetyl, benzoy], succinyl or diglycolyl, X2'S hydrogen, acetyl, benzoyl, succinyl or diglycolyl and Y is methyl.

   19. A compound according to claim 18 in which X, and X2 are hydrogen and Y is an isotopically labelled methyl group.

   20. A compound according to claim 19 in which Y is 11CH, "CH,, C11-1, and C'H, 21. A compound having the formula O.Y 2.

   1 v 1 1 0 v., wherein X, and X, which may be the same or different, are hydrogen or acyl and Y is alkyl or aryl.

   GB 2 127 023 A 15 22. A compound according to claim 21 in which X, and X2 are hydrogen and Y is methyl.

   23. A compound according to any one of claims 8 to 22 in which the asymetric center at carbon 24 has the (R) configuration.

   24. A compound according to any one of claims 8 to 22 in which the asymetric center at carbon 5 24 has the (S) configuration.

   25. A compound according to any one of claims 10, 12, 14 and 21 specifically identified herein.

   Printed for Her Majesty's Stationery Office by the Courier Press, Leamington Spa, 1984. Published by the Patent Office, 25 Southampton Buildings, London, WC2A 1 AY, from which copies may be obtained.